Bacteria of the oral, genital, respiratory, and gut microbiomes can exist as host- associated biofilm communities and biofilm-related infections are among the most serious diseases treated by clinicians. Bacterial associations and dynamics within the microenvironments of the biofilm structure are poorly understood on a global level. Global gene-expression dynamics of whole biofilms have been studied on the temporal scale, which lacks the resolution to decipher bacterial physiology and interactions within the microenvironments of biofilm. Thus, global gene-expression dynamics vary in two dimensions, space and time, which modulate the physiology of bacteria within their biofilm microenvironments. We have made recent advances in transcriptome analysis of single-cells have made fine-resolution functional genomic analysis of bacteria within their microenvironments possible. Our preliminary data of Pseudomonas aeruginosa (Pa) single-species biofilm has yielded many interesting aspects of this bacterium in its biofilm microenvironments and its organized spatial gene-expression pattern that make- up the living and functioning biofilm as a whole. Herein, we further proposed single- and mixed-species biofilm models of Pa and Burkholderia cenocepacia (Bc) to begin the process of unraveling the spatiotemporal global gene-expression pattern that contributes to the physiological- and interactions-dynamics of bacteria within their biofilm microenvironments. Aim 1 will determine the global gene-expression dynamics and physiology of Pa and Bc within mature single-species biofilms and Aim 2 will determine the global gene-expression dynamics, physiology, and interactions of Pa and Bc within mixed-species biofilms spatiotemporally in vitro, with validation in vivo. Upon completion of these studies, we expect to discover numerous answers to mysterious questions regarding the dynamics of bacterial physiology, interaction, and cooperation within microenvironments of single- and mixed-species biofilms at a global level. This will serve as a foundation and framework to further study the functional genomics of other host- associated biofilm communities and many important biofilm-related infections leading to novel treatment strategies.